The long-range goal of the proposed research is to understand the molecular mechanisms by which mutations in Myosin Binding Protein-C (MyBP-C) cause hypertrophic cardiomyopathy (HCM), an autosomal dominant disorder that affects 1 in 500 people and is the most common cause of sudden cardiac death in adolescents and young adults. Mutations in cardiac (c) MyBP-C are among the most frequent causes of HCM with >149 distinct mutations described so far. However, despite progress in identifying genetic causes of HCM, the molecular mechanism(s) by which any single cMyBP-C mutation causes disease are still unknown. Reduced amounts of cMyBP-C were recently reported in affected human myocardium, suggesting that loss of functional cMyBP-C from an affected allele (i.e., haploinsufficiency) is a common factor contributing to cardiac dysfunction. However, alternative possibilities that affected proteins impair contractile function or that processing and degradation of improperly folded proteins cause aberrant cell function have not been eliminated. These distinctions are critical for designing effective therapeutic strategies to overcome HCM, yet definitive evidence in support of the different possibilities has not been obtained in part because of the limited availability of human biopsies and in part because engineered mouse models do not fully recapitulate either the human disease phenotype or the proximal cell processes that lead to disease. Experiments proposed here will overcome these limitations by utilizing the only naturally occurring large animal model of HCM that both closely resembles the human disease phenotype and that has a known genetic cause. The mutation is a spontaneous missense mutation in cMyBP-C in domestic Maine Coon cats that results in a proline for alanine substitution at codon 31 (A31P). The mutation results in a single amino acid substitution, but causes an anomalous decrease in total amounts of cMyBP-C protein. Because similar decreases in cMyBP-C have been reported for missense cMyBP-C mutations in human myocardium, the feline A31P model offers a unique opportunity to distinguish between three primary factors proposed as causative in human disease i.e., dominant negative effects of a single amino acid point mutation, gene dosage effects, and cellular protein folding/trafficking defects. The proposed experiments will test the hypothesis that reduced amounts of cMyBP-C in sarcomeres (i.e., haploinsufficiency) of affected cats leads to contractile deficits that ultimately cause cardiac dysfunction. Specific aims of the project are to determine 1) the expression level and subcellular localization of total cMyBP-C protein in cats carrying the A31P mutation, 2) effects of the A31P mutation on myocyte contractile properties, and 3) effects of the A31P mutation on the myocardial ubiquitin-proteasome (UPS) system. Collectively, results from these studies will provide critical insights into molecular mechanisms by which mutations in cMyBP-C cause disease and will provide a significant and lasting impact on HCM research by developing a unique animal model that will be a resource for basic research and that will aid in the design and testing of therapeutic strategies for the treatment of cardiomyopathies linked to cMyBP-C. PUBLIC HEALTH RELEVANCE: The proposed studies will investigate basic pathogenic mechanisms by which the A31P mutation in the cardiac myosin binding protein-C (cMyBP-C) causes inherited hypertrophic cardiomyopathy (HCM) in Maine coon cats, a breed of domestic cat. In humans, HCM affects an estimated 1 in 500 people and is the leading cause of sudden death in adolescent and young adults. Because mutations affecting cMyBP-C are a leading cause of HCM in humans, it is anticipated that the proposed studies in Maine Coon cats will provide insights into human disease and will ultimately contribute to advances in disease diagnosis, prognosis, and treatment of HCM linked to cMyBP-C.